TIA-1 RRM23 binding and recognition of target oligonucleotides.

TIA-1 (T-cell restricted intracellular antigen-1) is an RNA-binding protein involved in splicing and translational repression. It mainly interacts with RNA via its second and third RNA recognition motifs (RRMs), with specificity for U-rich sequences directed by RRM2. It has recently been shown that RRM3 also contributes to binding, with preferential binding for C-rich sequences. Here we designed UC-rich and CU-rich 10-nt sequences for engagement of both RRM2 and RRM3 and demonstrated that the TIA-1 RRM23 construct preferentially binds the UC-rich RNA ligand (5΄-UUUUUACUCC-3΄). Interestingly, this binding depends on the presence of Lys274 that is C-terminal to RRM3 and binding to equivalent DNA sequences occurs with similar affinity. Small-angle X-ray scattering was used to demonstrate that, upon complex formation with target RNA or DNA, TIA-1 RRM23 adopts a compact structure, showing that both RRMs engage with the target 10-nt sequences to form the complex. We also report the crystal structure of TIA-1 RRM2 in complex with DNA to 2.3 Šresolution providing the first atomic resolution structure of any TIA protein RRM in complex with oligonucleotide. Together our data support a specific mode of TIA-1 RRM23 interaction with target oligonucleotides consistent with the role of TIA-1 in binding RNA to regulate gene expression.

Solution structure and DNA binding of the catalytic domain of the large serine resolvase TnpX.

The transfer of antibiotic resistance between bacteria is mediated by mobile genetic elements such as plasmids and transposons. TnpX is a member of the large serine recombinase subgroup of site-specific recombinases and is responsible for the excision and insertion of mobile genetic elements that encode chloramphenicol resistance in the pathogens Clostridium perfringens and Clostridium difficile. TnpX consists of three structural domains: domain I contains the catalytic site, whereas domains II and III contain DNA-binding motifs. We have solved the solution structure of residues 1-120 of the catalytic domain I of TnpX. The TnpX catalytic domain shares the same overall fold as other serine recombinases; however, differences are evident in the identity of the proposed hydrogen donor and in the size, amino acid composition, conformation, and dynamics of the TnpX active site loops. To obtain the interaction surface of TnpX1-120 , we titrated a DNA oligonucleotide containing the circular intermediate joint attCI recombination site into (15) N-labeled TnpX1-120 and observed progressive nuclear magnetic resonance chemical shift perturbations using (15) N HSQC spectra. Perturbations were largely confined to a region surrounding the catalytic serine and encompassed residues of the active site loops. Utilizing the perturbation map and the data-driven docking program, HADDOCK, we have generated a model of the DNA interaction complex for the TnpX catalytic domain.

The binding of TIA-1 to RNA C-rich sequences is driven by its C-terminal RRM domain.

T-cell intracellular antigen-1 (TIA-1) is a key DNA/RNA binding protein that regulates translation by sequestering target mRNAs in stress granules (SG) in response to stress conditions. TIA-1 possesses three RNA recognition motifs (RRM) along with a glutamine-rich domain, with the central domains (RRM2 and RRM3) acting as RNA binding platforms. While the RRM2 domain, which displays high affinity for U-rich RNA sequences, is primarily responsible for interaction with RNA, the contribution of RRM3 to bind RNA as well as the target RNA sequences that it binds preferentially are still unknown. Here we combined nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) techniques to elucidate the sequence specificity of TIA-1 RRM3. With a novel approach using saturation transfer difference NMR (STD-NMR) to quantify protein-nucleic acids interactions, we demonstrate that isolated RRM3 binds to both C- and U-rich stretches with micromolar affinity. In combination with RRM2 and in the context of full-length TIA-1, RRM3 significantly enhanced the binding to RNA, particularly to cytosine-rich RNA oligos, as assessed by biotinylated RNA pull-down analysis. Our findings provide new insight into the role of RRM3 in regulating TIA-1 binding to C-rich stretches, that are abundant at the 5' TOPs (5' terminal oligopyrimidine tracts) of mRNAs whose translation is repressed under stress situations.

Cooperative interplay of let-7 mimic and HuR with MYC RNA.

Both RNA-binding proteins (RBP) and miRNA play important roles in the regulation of mRNA expression, often acting together to regulate a target mRNA. In some cases the RBP and miRNA have been reported to act competitively, but in other instances they function cooperatively. Here, we investigated HuR function as an enhancer of let-7-mediated translational repression of c-Myc despite the separation of their binding sites. Using an in vitro system, we determined that a let-7 mimic, consisting of single-stranded (ss)DNA complementary to the let-7 binding site, enhanced the affinity of HuR for a 122-nt MYC RNA encompassing both binding sites. This finding supports the biophysical principle of cooperative binding by an RBP and miRNA purely through interactions at distal mRNA binding sites.